Integrating Anammox into Vegetated Biofiltration Systems for Enhanced Nitrogen Removal: Performance, Capacity, and Molecular Mechanisms

Vegetated biofiltration systems (VBS) are low-energy, nature-based solutions for water treatment; however, they often exhibit poor ammonium removal when treating ammonium-rich influents such as those encountered during overflow events or in decentralized applications. This study demonstrates that integrating anaerobic ammonium oxidation (anammox) into VBS significantly enhances the nitrogen removal performance and capacity. Over 25 weeks of operation, the anammox-integrated VBS (A-VBS) achieved substantially higher nitrogen removal efficiency (63.7 ± 1.8%) compared to the conventional VBS (C-VBS) (18.9 ± 3.0%) at a fixed hydraulic retention time (HRT) of 3 days. Enhanced performance persisted under shortened HRTs (48 and 24 h), indicating the hydraulic resilience of the anammox process. Microbial community analysis confirmed the establishment of the anammox-associated genus <i>Candidatus Brocadia</i> in A-VBS (1.2-2.2% relative abundance, along with a 4-fold increase in anammox 16S rRNA gene abundance), undetectable in C-VBS. ¹⁵N tracer assays showed that anammox contributed approximately 46.2% of N₂ production in A-VBS. Quantitative modeling revealed co-occurrence and synergistic interactions among nitrification (<i>amoA</i>, <i>nxrA</i>), anammox (anammox 16S rRNA), and denitrification (<i>narG, nirK</i>, <i>nirS</i>, <i>qnorB</i>, <i>nosZ</i>) pathways in driving total nitrogen removal. These findings suggest the potential of anammox-integrated VBS as an effective, sustainable solution for enhanced nitrogen management in urban water systems.